1. Field of the Invention
The invention in the fields of immunology and medicine is directed to a method for treating a category of neoplastic diseases that are manifest in sheaths surrounding organs (intrathecal) by administering tumoricidal superantigens such as bacterial enterotoxins and various biologically active derivatives thereof.
2. Description of the Background Art
Staphylococcal enterotoxins (“SE's”) are representative of a family of proteins known as “superantigens” (SAg)—the most powerful T lymphocyte mitogens known. They can activate between about 5 and about 30% or the total T cell population compared to the activation of 0.01% or fewer T cells by conventional antigens. Moreover, these enterotoxins elicit strong polyclonal proliferative responses at concentrations about 103-fold lower than other T cell mitogens. The most potent SE on a per weight basis, Staphylococcal enterotoxin A (SEA), stimulates human T cell proliferation (measured as DNA synthesis) at concentrations of as low as 10−13-10−16M.
SAg-activated T cells produce a variety of cytokines, including interferon-γ (IFNγ), various interleukins and tumor necrosis factor-α (TNFα) (Dohlsten et al., Int. J. Cancer 54:482-488 (1993).
SAgs also stimulate other cell populations involved in innate and adaptive immunity and contribute to anti-tumor immunity. For example, SE's engage the variable (V) region of the T cell receptor (TCR) chain on the exposed face of the pleated sheet and the sides of the MHC class II molecule (Kiting B L et al., Adv Immunol. 1993; 54:99-166). SAgs augment TH1 cytokine response by CD4+ cells while also activating cells of the NK, NKT and γ/δ T cell lineages. Cytotoxic action of NK cells is augmented by the IFNγ produced by SAg activated T cells (Morita et al., Immunity 14:331-44. (2001) D'Orazio J A et al., J Immunol. 154:1014-23 (1995).
SAgs induce tumor killing in vivo when given alone or when conjugated to tumor-specific antibodies (Terman U.S. Pat. No. 6,221,351; Dohlsten U.S. Pat. No. 5,858,363). They are also effective when employed ex vivo to induce the generation of tumor sensitized T cells that are then administered in the “adoptive therapy” of (e.g., MCA 205/207) tumors (Shu et al. J Immunol. 152: 1277-1288 (1994). SAg-transfected tumor cells can reduce metastatic disease in an established murine mammary carcinoma model (Pulaski et al., Cancer Res. 60: 2710-5 (2000).
In addition to these biological activities, the SE's share common physicochemical properties. They are heat stable, trypsin-resistant, and soluble in water and salt solutions, have similar sedimentation coefficients, diffusion constants, partial specific volumes, isoelectric points, and extinction coefficients. Prior to more recent discoveries of additional SE's, earlier-described SEs were divided into five serological types designated SEA, Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C (SEC), Staphylococcal enterotoxin D (SED) and Staphylococcal enterotoxin E (SEE), which exhibit striking structural similarities.
An SE is a single polypeptide chain of about 30 kDa. All SEs have a characteristic disulfide loop near the middle of the chain. SEA is a flat monomer consisting or 233 amino acids divided into two domains: domain I comprising residues 31-116 and domain II comprising residues 117-233 together with the amino tail of residues 1-30. The biologically active regions of the proteins are evolutionarily conserved and show a relatively higher degree of sequence homology/similarity. One region of striking amino acid sequence homology between SEA, SEB, SEC, SED, and SEE is located immediately on the C-terminal side of Cys-106 (in SEA). This conserved region is thought to be responsible for T cell activation. A second conserved homology region, at about residue 147, is believed to be responsible for emetic activity. This emesis-inducing region can be deleted from SE's through genetic engineering; such modified SE's are also useful therapeutics in accordance with this invention.
Sequence analysis of SEs and comparison with other bacterial toxins revealed SEA, SEB, SEC, SED, Staphylococcal toxic shock-associated toxin (TSST-1, also known as SEF), and the Streptococcal pyrogenic exotoxins (SpE's) share considerable nucleic acid and amino acid sequence similarity (Betley et al., J. Bacteriol. 170: 34-41 (1988)). Thus, the SEs belong to a family of evolutionarily related proteins.
SEs bind to MHC class II molecules and TCRs in a manner quite distinct from conventional antigens. SEs engage the V region of the TCR β chain (Vβ region) on an exposed face of the β pleated sheet. SEs engage the “sides” of MHC class II molecule rather than engaging the groove as do conventional antigens. In contrast to SEB and the SEC, which bind only to the MHC class II α chain, SEA, as well as SEE and SED, also interact with the MHC class II α chain in a zinc-dependent manner (Fraser J D et al., Proc. Natl. Acad. Sci. 89:5507-11 (1992)).
T cell recognition of SAgs, such as SEs, via the TCR Vβ region is independent of other TCR components and diversity elements. Single amino acid positions and regions important for SAg-TCR interactions have been defined. These residues are located in the vicinity of the shallow cavity formed between the two SE domains. (Lavoie P M et al., Immunol. Rev. 168: 257-269 (1999). Substitution of amino acid residue Asn23 in SEB by Ala has demonstrated the importance of this position in SEB/TCR interactions. This particular residue is conserved among all of the SE's and may constitute a common anchor position for SE interaction with TCR structures. Amino acid residues in positions 60-64 of SEA contribute to the TCR interaction as do the Cys residues forming the intramolecular disulfide bridge (Kappler J et al., J. Exp. Med. 175 387-96 (1992)). For SEC2 and SEC3, the key points of interaction in the TCR Vβ region are located in the CDR1, CDR2 and HRV4 regions of the TCR Vβ3 chain (Deringer J R et al., Mol. Microbiol. 22: 523-534 (1996)). Hence, multiple and highly variable parts of the Vβ region contribute to the formation of the TCRs SE binding site.
Thus far, no single, linear consensus motif in the TCR Vβ displaying a high affinity interaction with particular enterotoxins has been identified. A significant contribution of the TCRα chain in SE-TCR recognition is acknowledged (Smith et al., J. Immunol. 149: 887-896 (1992)). It is apparently the distinctive binding characteristics of SEs which bypass the highly variable parts of the MHC class II and TCR molecules that endows SEs with their ability to activate such a high frequency of T cells and cause massive proliferation, cytokine induction and cytotoxic T cell generation. These properties are shared by other proteins produced by various infectious agents. Together, these proteins form a well recognized family of molecules, Sags, because of their aforementioned biological effects.
Mycoplasmal, viral, and other bacterial proteins are SAgs. In addition to SEs and SpEs, examples include Yersinia pseudotuberculosis mitogenic protein (“YPM”), and Clostridium perfringens toxin A. All SAgs activate T cells without a requirement for conventional antigen processing, and the responding T cells do not respond in a conventional MHC restricted manner. As noted, SAgs bind to and evoke responses from all T cells expressing certain TCR Vβ gene products independently of other TCR structures. CD4− CD8− TCR α/β T cells and γ/δ T cells all respond to SAgs by proliferation, production of TH1 cytokines and generation of cytotoxic activity.
Native SEs are known to induce anti-tumor effects. Administration of SEB produced antitumor effects against established tumors in two animal species, rabbits and mice, with tumors of five different histologic types: rabbit VX-2 carcinoma (Terman et al., U.S. Pat. No. 6,126,945; Terman, U.S. Pat. No. 6,340,461), murine CL 62 melanomas (Penna C. et al., Cancer Res. 54: 2738-2743 (1994)), murine A/20 lymphoma (Kalland T. Declaration in U.S. Ser. No. 07/689/799 (1992)), murine PRO4L fibrosarcoma (Newell et al., Proc Natl. Acad. Sci. 88: 1074-1079 (1991)) and human SW 620 colon carcinoma (Dohlsten et al., Eur. J. Immunol. 21: 1229-1233 (1991)). In these studies, parenterally-administered SEB induced objective anti-tumor effects at primary and metastatic sites. SEB was used ex vivo to stimulate a population of T cells pre-exposed to tumor, which, upon re-infusion into host animals with established pulmonary metastases, induced a substantial reduction of metastases. SEB activated T cell anti-tumor effect was specific for the immunizing tumor; the SEB stimulated T cells produced IFNγ which was thought to be an important mediator of the anti-tumor effect (Shu S et al., J. Immunol. 152: 1277-88 (1994)).
Fusion polypeptides comprising SEA fused to a tumor specific monoclonal antibody (mAb), designated “SEA-mAb,” induced tumoricidal responses in the murine B16 melanoma model (Dohlsten M et al., Proc Natl Acad Sci 91:8945-9 (1994); Dohlsten M et al., Proc. Natl. Acad. Sci. 88:9287-91 (1991).
Because native SEA alone was found to be ineffective in such models, Dohlsten and colleagues (U.S. Pat. No. 5,858,363) stated that native SAg would be of “low value” particularly against MHC class II-negative tumors. When the fusion polypeptides SEA-mAb and one comprising a mutant SEA, “D227A-mAb” (these authors used ‘MoAb’ rather than ‘mAb’ for abbreviating ‘monoclonal antibody’) were given to human patients with advanced colon carcinoma, SEs reacted with preexisting “natural” SE-specific antibodies which diminished the antitumor effects in vivo. Following additional doses of SE-mAb preparations, the anti-SE antibody levels increased significantly (Giantonio et al., J. Clin. Oncol. 15:1994-2007 (1997); Alpaugh et al., Clin. Cancer Res. 4:1903-14 (1998); Persson et al., Adv. Drug Del. Res. 31: 143-152 (1998)). To date, efforts to overcome this problem have met with only partial success.
Tumors in Sheaths Encasing Organs
The appearance of tumors in sheaths (“theca”) encasing organs often results in production and accumulation of large volumes of fluid in the organs' sheath. Examples include (1) pleural effusion due to fluid in the pleural sheath surrounding the lung, (2) ascites originating from fluid accumulating in the peritoneal membrane and (3) cerebral edema due to metastatic carcinomatosis of the meninges. Such effusions and fluid accumulations generally develop at an advanced stage of the disease. Malignant pleural effusion (“MPE”) is the prototype of this condition. In the United States and Western Europe, 300,000 new cases of malignant pleural effusion are diagnosed annually (Antony V B et al., Eur. Respir. J. 18:402-419 (2001). This condition is caused by different types of tumors: lung cancer (35%), breast cancer (25%), lymphoma (10%), unknown primary malignancy (30%). It is the presenting manifestation in 10-50% of all cancers. When first evaluated, about 15% of lung cancer patients exhibit a pleural effusion. Fifty percent of cancer patients develop MPE at some point in their disease process, and up to 75% of MPE cases are symptomatic from their effusions upon presentation. The appearance of a pleural effusion in non small cell lung cancer (NSCLC) signifies Stage Mb or Stage IV and a poor prognosis with a median survival on the order to 2-3 months (1, 7-11). In this group, no significant difference in survival were observed between those with cytologically positive and negative effusions (12). Although most of these patients are symptomatic and/or disabled from their effusions, they are not surgical candidates. They are usually offered palliative treatment with chemical pleurodesis.
Malignant ascites is associated with 30-50% of ovarian tumors. Endometrial, breast, colonic, gastric and pancreatic carcinomas make up more than 80% or the tumors associated with intra-abdominal seeding of tumor cells and ascites formation. Ascites may be the presenting manifestation in 4-69% of cases.
The major therapies for MPE include talc poudrage, talc slurry, doxycycline and bleomycin instillation (Veena et al. Am J. Crit. Care Med. 162: 1987-2001 (2000)). These therapies require 3-12 days of hospitalization with EKG and oximetry monitoring. A chest tube is inserted, and the therapeutic agent is infused and allowed to distribute over the pleural membranes. The chest tube is then connected to closed negative-pressure water seal drainage until pleural fluid volume drops below 100 ml/24 hours. Respiratory therapy is usually given at least once daily.
Talc poudrage requires the use of operating room and general anesthesia for thoracostomy and talc insufflation, followed by recovery room observation. Talc induces respiratory complications in up to 33% of patients and acute respiratory distress and hypoxemia in 10% of patients. Response rates to bleomycin and doxycycline range between 50% and 70%, respectively and both require continuous chest tube drainage until the output is below 100 ml/24 hours. Indwelling pleural catheters for drainage and/or injection of a pleurodesis agent are an additional option (7,8); however, the catheter requires surgical placement followed by intermittent drainage of effusion fluid at home by the patient or a caregiver.
Intrapleurally administered agents or modalities that include (a) chemotherapeutic agents such as Cisplatin, Cytarabine, Doxorubicin fluorouracil, etoposide, and mitomycin C, (b) radiation and (c) biotherapeutic agents such as IL-2, various interferons, and bacterially derived immunostimulatory agents such as Corynebacterium parvum have been ineffective against MPEs. Thoracentesis or chest tube drainage alone results in recurrence rates of 98% and 85% respectively within 30 days. Intraperitoneal cisplatin and etoposide has produced a complete response rate of 30% of malignant ascites. However the only randomized study has failed to show any benefit for intraperitoneal therapy over conventional intravenous chemotherapy in the initial management of stage II C to IV ovarian cancer. No definitive success of various biologic agents, e.g., IFN-α, β, and γ, TNFα or IL-2 has been reported.
The present invention overcomes these deficiencies in the treatment of MPE by providing a new therapeutic approach to this manifestation of cancer. Unlike existing therapies, The present invention is carried out entirely in an outpatient setting and requires no hospitalization at a cost several hundred percent below that of existing therapy. Major costs of the other therapies originating from hospitalization, chest tube insertion, operating and recovery room expense, respiratory therapy and in-hospital chest tube drainage, are eliminated
Intratumoral SAg Therapy
Prior to the present invention, therapeutic uses of SAgs have been limited to systemic administration. To improve the ability SAgs to localize to a tumor, investigators have taken two approaches. In one approach, they have produced mutant SAg molecules with reduced binding to MHC class II molecules (Hansson J et al., Proc. Nail Acad. Acad Sci. USA 94: 2489-94 (1997)). In the second approach, they have conjugated a tumor specific antibody to the SAg (Dohlsten M et al., Proc Nail Acad Sci USA 91:8945-9 (1994); Dohlsten M et al., Proc Nail Acad Sci USA 88:9287-91 (1991)). However, because SAg-specific antibodies are found in all humans, these engineered molecules, rather than localizing to tumors, are more likely to be directed to reticuloendothelial tissues where they are degraded and eliminated. The researchers cited above expressly asserted (U.S. Pat. No. 5,858,363 that native SAgs would be of “low value” for such antitumor therapy because cells of most clinically important tumors do not express MEM class II molecules. It is therefore evident that those working in this field, led by the investigators cited above, did not envision the use of the SAgs by intratumoral administration. In contrast to systemic administration, intratumoral delivery of a SAg would not require alteration of the native molecule and, as conceived by the present inventor, the presence of natural antibodies throughout the body can actually assist intratumorally-administered SAgs in evoking a tumoricidal response.